Nucleic acid probe hybridizations (DNA or RNA probe hybridizations) require biological samples to be processed to provide sample or target nucleic acid prior to hybridization with extrinsic nucleic acid probes. This processing is required because in order to hybridize the nucleic acid probes with complementary portions of the sample nucleic acids in turn contained within microorganisms defined as fungal cells, bacterial cells or viral particles, the sample nucleic acid must be released from inside the structure so as to be rendered accessible to the probes.
Nucleic acids have been traditionally released from biological systems via a variety of methods including the chemical action of detergents, bases, acids, chaotropes, organics and mixtures of these chemicals on samples. Various organisms, cells, bacteria or viruses characteristically require different chemical conditions in order to effectively release their nucleic acids. Physical methods of processing samples have also been practiced and include pressure, heat, freeze-thaw cycles and sonication with and without glass beads. Further, combinations of physical and chemical methods have also been used to prepare samples for DNA probe hybridizations such as chemical cell lysis followed by sonication.
Sonication devices employ ultrasonic vibrations and have previously been employed for a variety of processes including homogenization, cellular disruption, molecular disassociation, humidification, aerosol generation, lubrication, coating systems and instrument nebulizers. Ultrasound is commonly understood to encompass the propagation of a sound wave in a solution with the accompanying formation of regions of compression and rarefaction. The alternating acoustic pressure causes the making and breaking of microscopic bubbles. Pressure changes of 20,000 atmospheres can be achieved in cavitational microenvironments. The microscopic bubbles or cavities grow over many cycles and collapse with great force once they reach certain critical dimensions known as the critical bubble size. The critical bubble size is substantially a function of frequency; as frequency is increased, more power is required in order to produce cavitation. Above 1 MHz, the intensity of sonication is greatly diminished and cavitation cannot be produced at all above 2.5 MHz.
A number of different sonicators which cause cavitation have been used in studies involving nucleic acids including systems offered commercially by Heat Systems Ultrasonics, Tomy Co., Rapidis, Raytheon, Mullard and Branson. These commercial sonicators have traditionally been designed to resonate at a frequency between 5 and 35 KHz and most resonate at approximately 20 KHz. The typical commercially available sonicator has been used in one of two modes: (1) direct immersion of the vibrating probe into the sample, and (2) placement of both the container holding the sample and the vibrating transducer of the sonication unit into a common liquid or bath. The first mode disadvantageously incurs sample-to-sample carry-over and thus is not practical in a clinical setting. In the second mode of use, the liquid acts to couple the sonic vibrations to the sample in the container or cuvette and may also assist in cooling and/or controlling the sample temperature. While this method is relatively efficient, it is disadvantageously complicated by the necessary mechanics of the liquid bath and the contamination threat posed thereby. Accordingly its application in the clinical environment is also limited.
It is an object of the present invention to provide a new sonication system having an effectiveness comparable to the liquid bath sonication method.
It is another object of the present invention to provide a new sonication method for preparing samples for DNA probe hybridization which avoids the contamination disadvantages associated with immersion type sonication.
It is known that when a conventional ultrasonic transducer is applied directly to the surface of a container, ultrasonic energy is not readily transmitted to a liquid contained within the container. This occurs because a significant percentage of the energy is lost in the form of heat, either in the contact surfaces of the transducer and the container wall, or in the container material itself. The amount of energy actually transmitted is further limited by the acoustic impedances of each material. Thus, only a relatively small amount of the initial energy is actually transmitted to the liquid and cavitation fails to occur unless a very large excess of initial energy is applied. Application of such an excess of energy is generally highly disadvantageous because such results in highly localized heating often to the extent that melting of the container may occur. Such a result is quite clearly unacceptable, particularly in a clinical environment where samples may be dangerously infective.
It is yet another object of the present invention to provide a new method for preparing samples for DNA hybridization which utilize direct transducer - container contact while avoiding the application of previously required high levels of energy.